Analysis of related factors of CRA in lung cancer patients with different serum iron levels: A retrospective cohort study

Abstract Background Serum iron, an essential component of hemoglobin (Hb) synthesis in vivo, is a crucial parameter for evaluating the body's iron storage and metabolism capacity. Iron deficiency leads to reduced Hb synthesis in red blood cells and smaller red blood cell volume, ultimately resulting in iron‐deficiency anemia. Although serum iron cannot independently evaluate iron storage or metabolism ability, it can reflect iron concentration in vivo and serve as a good predictor of iron‐deficiency anemia. Therefore, exploring the influence of different serum iron levels on anemia and diagnosing and treating iron deficiency in the early stages is of great significance for patients with lung cancer. Aim This study aims to explore the related factors of cancer‐related anemia (CRA) in lung cancer and construct a nomogram prediction model to evaluate the risk of CRA in patients with different serum iron levels. Methods A single‐center retrospective cohort study was conducted, including 1610 patients with lung cancer, of whom 1040 had CRA. The relationship between CRA and its influencing factors was analyzed using multiple linear regression models. Lung cancer patients were divided into two groups according to their serum iron levels: decreased serum iron and normal serum iron. Each group was randomly divided into a training cohort and a validation cohort at a ratio of 7:3. The influencing factors were screened by univariate and multivariate logistic regression analyses, and nomogram models were constructed. The area under the receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the models. Results CRA in lung cancer is mainly related to surgery, chemotherapy, Karnofsky Performance Status (KPS) score, serum iron, C‐reactive protein (CRP), albumin, and total cholesterol (p < 0.05). CRA in lung cancer patients with decreased serum iron is primarily associated with albumin, age, and cancer staging, while CRA in lung cancer patients with normal serum iron is mainly related to CRP, albumin, total cholesterol, and cancer staging. The area under the ROC curve of the training cohort and validation cohort for the prediction model of lung cancer patients with decreased serum iron was 0.758 and 0.760, respectively. Similarly, the area under the ROC curve of the training cohort and validation cohort for the prediction model of lung cancer patients with normal serum iron was 0.715 and 0.730, respectively. The calibration curves of both prediction models were around the ideal 45° line, suggesting good discrimination and calibration. DCA showed that the nomograms had good clinical utility. Conclusion Both models have good reliability and validity and have significant clinical value. They can help doctors better assess the risk of developing CRA in lung cancer patients. CRP is a risk factor for CRA in lung cancer patients with normal serum iron but not in patients with decreased serum iron. Therefore, whether CRP and the inflammatory state represented by CRP will further aggravate the decrease in serum iron levels, thus contributing to anemia, warrants further study.


| INTRODUCTION
Lung cancer is one of the most common malignant tumors, with an incidence second only to breast cancer and the highest mortality rate among all solid tumors worldwide. 1The five-year survival rate remains below 20%. 2,3CRA is a common complication of tumors that is often overlooked.Its main manifestations are decreased red blood cells, hemoglobin (Hb) concentration, or red blood cell volume in peripheral blood.CRA is also common in lung cancer patients, with epidemiological investigations revealing an incidence as high as 32.09%. 4he occurrence and development of CRA seriously affect the prognosis of lung cancer patients.Studies have shown that CRA has become an independent risk factor endangering the survival rate and quality of life of lung cancer patients. 5Tumors can induce anemia and anemia can lead to hypoxia in tumor cells, induce changes in tumor gene expression, and inhibit tumor cell apoptosis, thereby enhancing their invasion ability and leading to the failure of antitumor treatment. 6,7Additionally, CRA can cause various related symptoms, 8 which seriously affect the efficacy of antitumor treatment and patients' quality of life.Current studies show that CRA in lung cancer is closely related to cancer staging, type, disease course, and therapeutic regimen, 9,10 but a risk factor prediction model is lacking.
Iron deficiency, which has a high incidence, is a common complication of tumors and an accompanying symptom of CRA.Iron deficiency is a continuous spectrum ranging from iron storage deficiency to irondeficient erythropoiesis, which eventually leads to iron-deficiency anemia. 11Serum iron is an important component of Hb synthesis in the body, representing the iron concentration and occupying an indispensable position in iron deficiency.In the past, iron deficiency was thought to be related to malnutrition and was given more attention in digestive tract tumors.However, few studies have been conducted on CRA in lung cancer, with most studies limited to exploring its influencing factors and no effective prediction model established.Therefore, the significance of iron content represented by serum iron in the formation of CRA in lung cancer remains unclear.Based on univariate and multivariate logistic regression analyses of CRA-related factors in lung cancer, this study established nomogram models to predict the CRA of decreased and normal serum iron levels in lung cancer to provide a scientific basis for clinical treatment.

| Research object
This study is a retrospective cohort study.From September 2017 to August 2022, patients with lung cancer were collected from the oncology department of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine staging, while CRA in lung cancer patients with normal serum iron is mainly related to CRP, albumin, total cholesterol, and cancer staging.The area under the ROC curve of the training cohort and validation cohort for the prediction model of lung cancer patients with decreased serum iron was 0.758 and 0.760, respectively.Similarly, the area under the ROC curve of the training cohort and validation cohort for the prediction model of lung cancer patients with normal serum iron was 0.715 and 0.730, respectively.The calibration curves of both prediction models were around the ideal 45° line, suggesting good discrimination and calibration.DCA showed that the nomograms had good clinical utility.

| RESULTS
A total of 1610 patients with lung cancer were included.According to the inclusion and exclusion criteria, 1040 patients with lung cancer CRA were included (64.6%), including 390 patients with decreased serum iron (37.5%) and 650 patients with normal serum iron (62.5%).Among the 1610 patients with lung cancer, there were 458 patients with decreased serum iron (28.45%) and 1152 patients with normal serum iron (71.55%).See Figure 1. 3.1 | CRA analysis of lung cancer   4.

| Analysis of related factors with decreased serum iron in lung cancer patients
Univariate analysis shows that the differences in whether age, sex, different cancer staging, surgery, and albumin will cause the occurrence of CRA in lung cancer patients with decreased serum iron have statistical significance.Taking the results of univariate analysis with p < 0.01 to select affecting factors as the independent variable and whether CRA occurs or not as the dependent variable, multivariate logistic regression is used to analyze the related factors of the occurrence of CRA in lung cancer patients with decreased serum iron to establish a logistic regression prediction model.The overall prediction accuracy of the model is 86.2%.The results show that whether CRA occurs or not in lung cancer patients with decreased serum iron is mainly related to albumin, age, and cancer staging (p < 0.05).See Table 5.

| Analysis of factors in patients with normal serum iron levels in lung cancer
Univariate analysis revealed statistically significant differences in the impact of age, gender, cancer staging, surgery, radiation therapy, CRP, albumin, and total cholesterol on the incidence of CRA in lung cancer patients with normal serum iron levels.Using a significance threshold of p < 0.01, factors identified through univariate analysis were selected as independent variables, with the incidence of CRA as the dependent variable.Multivariate logistic regression analysis was then employed to examine the factors influencing CRA incidence among these patients, culminating in the development of a logistic regression prediction model.This model exhibited an overall prediction accuracy of 68.2%.Analysis indicated that CRP, albumin, total cholesterol, and cancer staging were primarily associated with CRA incidence in lung cancer patients with normal serum iron levels (p < 0.05).Refer to Table 6 for detailed information.

| Development of a nomogram prediction model
The influencing factors identified through the analysis of lung cancer patients with either decreased or normal serum iron levels were utilized to create nomogram prediction models for CRA incidence in these two patient groups.In each nomogram, variables were assigned scores on a top score line.The total score, derived by summing the scores of all variables, corresponds to the predicted probability of anemia.See Figure 2 for the nomograms.3C,D).Comparison of ROC curves for both models demonstrated their effective discriminative power.
Calibration curves indicated that both prediction models were well-calibrated (refer to Figure 4).Additionally, the net benefit of clinical intervention based on the nomogram models surpassed that of the nointervention approach, indicating the models' significant clinical efficacy (refer to Figure 5).

| DISCUSSION
CRA is a prevalent complication of malignant tumors that significantly impacts a patient's prognosis.At  present, there are few studies on the influencing factors of CRA in lung cancer.Therefore, it is crucial to actively investigate the related influencing factors of CRA to prevent and treat the disease efficiently.In this study, the influencing factors were screened by univariate and multivariate logistic regression analyses, and nomogram models were constructed to evaluate the risk of CRA in patients with different serum iron levels in lung cancer.We found that lung cancer CRA is primarily related to the patient's past surgeries, chemotherapy, KPS score, serum iron, CRP, albumin, and total cholesterol levels through the univariate analysis of clinical data and laboratory indices of 1040 lung cancer patients with CRA.Surgery can lead to anemia due to the trauma associated with lung cancer surgery, intraoperative bleeding, and other factors that increase the likelihood of anemia. 14hemotherapy is another common cause of anemia, as it can lead to bone marrow suppression and cause gastrointestinal reactions such as nausea and vomiting, resulting in anemia. 15The KPS score is used to assess a patient's general health condition and forms the basis for their antitumor treatment plan.Malignant tumors are high-consumption and high-metabolic diseases that lead to malnutrition, resulting in low serum albumin, total cholesterol levels, and a decrease in raw materials for erythropoiesis such as iron, vitamin B12, and folic acid, thus leading to anemia.Additionally, as the malignancy progresses, patients experience various accompanying symptoms, leading to an inflammatory state in the body and an increase in inflammatory factors.Tumor-related inflammation may interfere with nutrition and hamper hematopoiesis, leading to anemia. 16,17t is worth noting that iron deficiency is a common cause of anemia in patients with malignant tumors.To evaluate iron deficiency, ferritin and transferrin saturation are often used in clinical settings, with ferritin being the most sensitive laboratory index.In general, the lower the ferritin level, the higher the chances of having irondeficiency anemia.However, in specific situations, such as inflammation or malnutrition, ferritin may falsely increase, 18,19 seriously impacting the diagnosis of iron deficiency and consequently affecting anemia.Therefore, it is crucial to explore the impact of serum iron on anemia actively.1][22] Although it cannot evaluate the body's iron storage or metabolism ability, it can reflect the iron concentration in vivo.When iron metabolism and homeostasis are disrupted in patients with malignant tumors, red blood cells may suffer from insufficient iron supply, leading to anemia. 23Iron deficiency leads to reduced Hb synthesis in red blood cells, smaller red blood cell volume, and decreased oxygen-carrying capacity, ultimately resulting in iron-deficiency anemia. 24Although serum iron cannot serve as the diagnostic standard for iron deficiency, it can be a good predictor of iron-deficiency anemia.Therefore, determining the effect of different serum iron levels on CRA in lung cancer patients and diagnosing and treating iron deficiency in early stages may be critical in managing CRA effectively.
This study utilizes multivariate logistic regression analysis to identify the related influencing factors that affect the occurrence of CRA in lung cancer patients with decreased serum iron, which include albumin, age, and cancer staging.Additionally, the related influencing factors for patients with normal serum iron are found to be CRP, albumin, total cholesterol, and cancer staging.Based on these factors, nomogram prediction models for CRA are constructed for both patient groups, demonstrating good prediction efficiency and clinical practicality.Previous studies have shown that serum iron levels are negatively correlated with tumor staging and positively correlated with patient prognosis in lung cancer cases, 25 as the incidence of CRA in lung cancer patients tends to increase with later stages of cancer and lower levels of serum iron due to tumor cell proliferation and increased demand for iron.Malignant tumors are consumptive diseases, and with the progression of the disease, some patients with advanced lung cancer consume more in the body because of the deterioration of malignant fluid, which leads to the decrease in serum iron content of hematopoietic raw materials, thus leading to CRA.Moreover, different pathological types are also closely related to disease progression.For example, adenocarcinoma is prone to distant metastasis, and squamous carcinoma is prone to hemoptysis, and disease progression will also aggravate anemia. 26Therefore, the degree of disease progression can also be used as an independent risk factor for CRA. 27Furthermore, age is also identified as an important risk factor for CRA among lung cancer patients, with elderly patients being more susceptible due to decreased hematopoietic raw material absorption, erythropoietin (EPO) secretion, and bone marrow erythropoiesis. 28,29t is of great significance to evaluate the effects of serum albumin, total cholesterol, and CRP on serum iron.Serum albumin and total cholesterol are effective indicators to evaluate the nutritional status of the body, and the decrease in their levels reflects malnutrition.CRP can be used as one of the most valuable indicators to evaluate the inflammatory state of cancer patients, 30 and its elevated level reflects the inflammatory state of the body.Iron deficiency in lung cancer is mainly manifested in the decrease of serum iron level, which is related to the decrease in iron storage and iron metabolism disorder.The decrease in iron storage is closely related to malnutrition, digestive system surgery, and chronic blood loss, and iron metabolism disorder is mainly related to chronic inflammation. 31he findings of this study further demonstrate that CRP is a significant influencing factor for CRA in patients with normal serum iron in lung cancer, which means that the inflammatory state represented by CRP can affect CRA.Studies have confirmed that when the body is in an inflammatory state, the release of inflammatory factors can be regarded as an important cause of CRA, 32 which mainly shows that proinflammatory cytokines can promote the secretion of inflammatory factors, such as interleukin-1 (IL-1), interleukin-6 (IL-6), interferon, and tumor necrosis factor (TNF), etc., which can not only inhibit the production of red blood cells but also promote their apoptosis, thus leading to inflammatory anemia or chronic disease. 33,34At the same time, the release of inflammatory cytokines is related to the production of reactive oxygen species, which can inhibit the production of red blood cells, interfere with nutritional status, and reduce the raw materials for red blood cell production, such as iron, vitamin B12, and folic acid, thus aggravating anemia. 17Moreover, reactive oxygen species can inhibit the immune response mediated by CD8+ T cells, which can contribute to the occurrence of anemia. 35RP is a risk factor that can cause the occurrence of CRA in lung cancer patients with normal serum iron; however, CRP has no significant effect on CRA in lung cancer patients with decreased serum iron, which may suggest that serum iron has a unique position in the process of CRA caused by inflammation.Previous studies have shown that CRP levels in patients with lung cancer are negatively correlated with serum iron levels.CRP can stimulate the synthesis of ferritin and promote its combination with intracellular iron, thus reducing the serum iron content in the body and increasing the risk of CRA and iron deficiency.36 In addition, the increase in CRP means that the body is in an inflammatory state, and inflammatory cytokines can stimulate the liver to synthesize hepcidin, 37 which can not only bind with iron transportrelated proteins on the surface of duodenal cells to weaken its effect of transferring iron but also regulate the transport process of iron through intestinal mucosa, inhibit the absorption of iron and the release of iron in cells, resulting in iron metabolism disorder and eventually leading to anemia.[38][39][40] The above results show that CRP and the inflammatory state represented by CRP can lead to a decrease in serum iron and further contribute to anemia.
However, in patients with lung cancer whose serum iron level has already decreased, the iron metabolism and iron homeostasis in the body have been disordered.Considering the dynamic balance and negative feedback mechanism of the body, whether CRP and the inflammatory state represented by CRP will further aggravate the decrease in serum iron levels, thus affecting anemia, has no research to support at present and is worth further discussion.
Since this study is a single-center and retrospective study with a relatively small sample size, the established model has not been verified in other institutions, and there may be some bias.Therefore, it is necessary to carry out larger sample size and multicenter, prospective studies in the future to further improve the results of this study and guide clinical treatment.

| CONCLUSION
In summary, chemotherapy, KPS score, serum iron, CRP, albumin, and total cholesterol are all factors related to CRA in lung cancer patients.Specifically, albumin, age, and cancer staging are related factors that impact the occurrence of CRA in lung cancer patients with decreased serum iron.On the other hand, CRP, albumin, total cholesterol, and cancer staging are factors that impact the occurrence of CRA in lung cancer patients with normal serum iron.We also discovered that CRP is a risk factor for the development of CRA in lung cancer patients with normal serum iron, but it is not a contributing factor for the reduction of CRA in patients with decreased serum iron levels.Therefore, whether CRP and the inflammatory state represented by CRP will further aggravate the decrease in serum iron levels, thus contributing to anemia, warrants further study.

:
Both models have good reliability and validity and have significant clinical value.They can help doctors better assess the risk of developing CRA in lung cancer patients.CRP is a risk factor for CRA in lung cancer patients with normal serum iron but not in patients with decreased serum iron.Therefore, whether CRP and the inflammatory state represented by CRP will further aggravate the decrease in serum iron levels, thus contributing to anemia, warrants further study.K E Y W O R D S cancer-related anemia, influencing factors, lung cancer, nomogram model, serum iron | 3 of 15 LI et al. affiliated with Shanghai University of Traditional Chinese Medicine. 2.1.1 | Diagnostic criteria The anemia standard refers to the World Health Organization's anemia classification standard in the Clinical Practice Guide of Tumor-related Anemia (2022 edition) published by the China Society of Clinical Oncology (CSCO) 12 : Grade 0 (Normal value): adult males should not have an Hb level of less than 130 g/L, nonpregnant adult females should not have an Hb level of less than 120 g/L, and pregnant adult females should not have an Hb level of less than 110 g/L; Grade 1 (Mild anemia): 110 g/L-Normal value; Grade 2 (Moderate anemia): 80 g/L-109 g/L; Grade 3 (Severe anemia): < 80 g/L.According to the Standard of Diagnosis and Curative Effect of Hematological Diseases, serum iron levels <8.95 μmol/L indicate decreased serum iron levels.

3. 2 |
Analysis of related factors of decreased serum iron and normal serum iron patients in lung cancer and construction of nomogram prediction model 3.2.1 | Clinical features A total of 458 patients (28.45%) with decreased serum iron levels in lung cancer are randomly divided into 7:3, including 325 patients in the training cohort (70.96%) and 133 patients in the validation cohort (29.04%).Thousand one hundred and fifty-two patients with normal serum iron levels in lung cancer (71.55%) are randomly divided into 7:3, including 843 patients in the training cohort (73.18%) and 309 patients in the validation cohort (26.82%).See Table

4
Clinical Characteristics of Lung Cancer Patients with Decreased Serum Iron.

F I G U R E 2
Nomogram prediction model of CRA in patients with decreased serum iron and patients with normal serum iron in lung cancer.(A) Nomogram prediction model of CRA in lung cancer patients with decreased serum iron.(B)nomogram prediction model of CRA in lung cancer patients with normal serum iron.F I G U R E 3 ROC curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron and normal serum iron.(A) Training Cohort ROC curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(B) Validation cohort ROC curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(C) Training cohort ROC curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.(D) Validation cohort ROC curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.

F I G U R E 4
Calibration curve of the nomogram prediction model about the occurrence of lung cancer patients with decreased serum iron and normal serum iron.(A) Training cohort calibration curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(B) Validation cohort calibration curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(C) Training cohort calibration curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.(D) Validation cohort calibration curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.

F I G U R E 5
DCA curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron and normal serum iron.(A) Training cohort DCA curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(B) Validation cohort DCA curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with decreased serum iron.(C) Training cohort DCA curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.(D) Validation cohort DCA curve of the nomogram prediction model about the occurrence of CRA in lung cancer patients with normal serum iron.LI et al. 13

Table 1 .
staging, pathological types, surgery, chemotherapy, and targeted therapy on different degrees of CRA are statistically different.Taking the KPS score equal to 70 as the boundary, the influence of KPS on different degrees of anemia is statistically different.See significance.Collinearity test: VIF <5, indicating that the results are accurate and reliable.The results show that CRA in lung cancer is mainly related to serum iron, CRP, albumin, total cholesterol, KPS score, surgery, and chemotherapy (p < 0.05).See Table3.
Analysis of Related Factors of CRA in Lung Cancer.
T A B L E 1 nomogram predicting CRA incidence in lung cancer patients with normal serum iron, areas under the ROC curve were calculated, resulting in values of 0.715 and 0.730 for the training and validation cohorts, respectively (refer to Figure Anemia of CRA Patients with Lung Cancer and Related Laboratory Examination Indicators.Multiple linear regression analysis of CRA related factors in lung cancer.
The clinical usefulness of the nomogram prediction models was assessed using DCA.DCA results showed that the risk thresholds for the training cohorts of the nomogram models predicting CRA incidence in lung cancer patients with decreased and normal serum iron were 0.3-0.85 and 0.3-0.8,respectively.For the validation cohorts, the risk thresholds were 0.3-0.95 and 0.3-0.83,respectively.T A B L E 2 **p < 0.01.T A B L E 3 *p < 0.05.**p < 0.01.

with decreased serum iron in lung caner Patients with normal serum iron in lung cancer Training cohort (n = 325) Validation cohort (n = 133) Training cohort (n = 843) Validation cohort (n
Analysis of related factors in patients with reduced serum iron Levels in lung cancer.
T A B L E 5 *p < 0.05.**p < 0.01.T A B L E 6 Analysis of related factors in patients with normal serum iron in lung cancer.*p < 0.05.**p < 0.01.